Short-Chain Polyphosphates Induce Tau Fibrillation and Neurotoxicity in Human iPSC-Derived Retinal Neurons

Short-Chain Polyphosphates Induce Tau Fibrillation and Neurotoxicity in Human iPSC-Derived Retinal Neurons | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Short-Chain Polyphosphates Induce Tau Fibrillation and Neurotoxicity in Human iPSC-Derived Retinal Neurons Paola Baiocco, Lorenzo Barolo, Lorenza Mautone, Ylenia Gigante, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5409787/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 May, 2025 Read the published version in Cell Death & Disease → Version 1 posted 9 You are reading this latest preprint version Abstract The onset of Alzheimer’s Disease and Frontotemporal Dementia is closely associated with the aggregation of tau, a multifunctional protein essential for neuronal stability and function. Given the role of tau aggregation in neurodegeneration, understanding the mechanisms behind its fibrils formation is crucial for developing therapeutic interventions to halt or reverse disease progression. However, the structural complexity and diverse aggregation pathways of tau present significant challenges, requiring comprehensive experimental studies. In this research, we demonstrate that short-chain polyphosphates, specifically sodium tripolyphosphate (NaTPP), effectively induce tau fibril formation in vitro using the microtubule-binding domain fragment (K18). NaTPP-induced fibrils display unique structural characteristics and aggregation kinetics compared to those induced by heparin, indicating distinct pathogenic pathways. Through molecular dynamics simulations, we show that NaTPP promotes aggregation by exposing key residues necessary for fibril formation, which remain concealed under non-aggregating conditions. This interaction drives tau into an aggregation-prone state, revealing a novel mechanism. Furthermore, our study indicates that human pluripotent stem cell-derived retinal neurons internalize NaTPP-induced fibrils within 24 hours, pointing to a potential pathway for tau spread in neurodegeneration. To explore the translational implications of NaTPP-induced fibrils, we assessed their long-term effects on cellular viability, tubulin integrity, and stress responses in retinal neuron cultures. Compared to heparin, NaTPP promoted fewer but longer fibrils with initially low cytotoxicity but induced a stress response marked by increased endogenous tau and p62/SQSTM1 expression. Prolonged exposure to NaTPP-induced oligomers significantly increased cytotoxicity, leading to tubulin fragmentation, altered caspase activity, and elevated levels of phosphorylated pathological tau. These findings align with a neurodegenerative phenotype, highlighting the relevance of polyphosphates in tau pathology. Overall, this research enhances our understanding of the role of polyphosphate in tau aggregation, linking it to key cellular pathways in neurodegeneration. Biological sciences/Neuroscience/Stem cells in the nervous system Biological sciences/Biochemistry/Proteins Figures Figure 1 Figure 2 Figure 3 Figure 4 INTRODUCTION Tau is a microtubule-associated protein primarily found in neurons, playing a critical role in maintaining axonal structural integrity ( 1 ),( 2 ). Its dysregulation leads to neurofibrillary tangles (NFTs), protein aggregates characteristic of neurodegenerative diseases such as Alzheimer’s disease (AD) and frontotemporal dementia (FTD) ( 3 ), (4), ( 5 ). In pathological conditions, tau undergoes conformational changes, transitioning from monomers to oligomers and eventually mature filaments ( 6 ), ( 7 ). The neurotoxicity is primarily associated with tau oligomers, which accumulate intracellularly during fibrils formation and spread between neurons, contributing to disease progression ( 8 ), ( 9 ), ( 10 ), ( 11 ). Recent studies have demonstrated that tau oligomers generated in vitro can be internalized by human neurons derived from induced pluripotent stem cells (iPSCs) and murine models ( 12 ), ( 13 ), ( 14 ). Once internalized, these oligomers act as templates, seeding the aggregation of endogenous tau and triggering a cascade that accelerates fibril formation, thereby enabling the long-term observation of neurodegenerative processes ( 15 ), ( 16 ), ( 17 ), ( 18 ). To model these aggregation dynamics, the microtubule-binding repeat region (MTBR), or K18 domain ( 19 ),( 20 ),( 21 ) is typically incubated with polyanionic compounds like polyunsaturated fatty acids, RNA, and polyglutamate ( 3 ), ( 22 ), ( 23 ), ( 24 ), ( 25 ). Heparin is frequently used for this purpose, but its use presents significant limitations, as it does not participate in tau fibrillation in vivo, and its polymorphic nature complicates the study of tau behavior under physiological conditions ( 22 ), ( 26 ). Polyphosphates (polyPs) have emerged as promising physiologically relevant alternatives for inducing tau fibrillation. Present in the extracellular space of the brain, polyPs play roles in cellular energy homeostasis, inflammation, and cell signaling ( 27 ),( 28 ), ( 29 ), ( 30 ), ( 31 ), ( 32 ),( 33 ),( 34 )( 35 ). More importantly, polyPs compete with tubulin for binding tau, promoting its aggregation into amyloid fibrils and potentially contributing to AD pathology ( 28 ), ( 29 ), ( 35 ). Tau fibrils formed with polyPs exhibit reduced cytotoxicity, especially in the early stages of aggregation, likely due to the reduced presence of toxic oligomers and protofibrils ( 36 ), ( 37 ), ( 38 ),( 39 ). Here, we report for the first time the possibility of obtaining mature fibrils of the K18 domain through a naturally occurring cofactor sodium tripolyphosphate (NaTPP) (Fig. 1 A-B). Our findings revealed a fundamental difference in aggregation kinetics and the number and length of filaments compared to heparin, enlightening a significant difference in the transition from smaller oligomeric species to longer-growing fibrils. Molecular dynamics simulations of K18 enlighten the exposure of three residues most involved in the fibril interface (GGG). The computational study shows clear compaction of K18 that, only in the presence of NaTPP, does an ensemble of conformations with a clear solvent exposure of the region involved in the tau fibril interface. A detailed analysis of protein-ligand contacts highlighted a set of key K18 residues, whose intermolecular contacts with NaTPP would ensure a stable area accessible to the solvent of the GGG patch, making the K18 conformation prone to binding with itself. The role of K18-NaTPP-induced aggregates was studied in vitro using human iPSC differentiated into retinal neurons. K18-NaTPP fibrillar structures, at lower incubation time, are generally reported as less cytotoxic than heparin, triggering a cellular protective response in the short term. However, this condition dramatically changed over a longer period, leading to the activation of the apoptotic pathway. Based on these findings, short polyphosphates may act as a potential catalyst for internalizing preformed K18-fibrils into iPSC retinal neurons, promoting tau-related conditions compatible with a pathological environment, including neurodegenerative diseases like AD and FTD. MATERIALS AND METHODS 1. Tau protein construct design, expression and purification This study used the K18 domain referred to as the MTBR domain of tau isoform 0N4R (residues 244 to 372) (Fig. 1 A) containing a point mutation C291S ( 40 ), ( 41 ), ( 42 ). Protein production and purification are described in SI ( 43 ), ( 44 ). The conformational assembly of K18 was analyzed by size exclusion chromatography using a HiLoad™ 26/600 Superdex 75 (Cytiva). 2. Formation and Fluorescence analysis of K18 fibrils K18 100 µM in PBS was reduced using 1 mM TCEP 10 minutes at 55°C. Sodium heparin or NaTPP was added in a 1:1 ratio to initiate tau protein aggregation in vitro . The samples (K18-Hep and K18-NaTPP) were kept at 37°C under rotation at 150 rpm for several time points. The fibrillation kinetics was analyzed using the BODIPY-based probe BT1, loaded into humanized ferritin nanocages due to the limited specificity of thioflavin T (ThT) towards K18 fibrils ( 45 ), ( 46 ), ( 47 ). All the measurements were made by using the RF-6000 fluorimeter (Shimadzu RF-6000). Further details and the preparation of K18 fibrils for STEM imaging are given in SI. 3. Computational Methods For each of the molecular systems considered in this work, we used Gromacs 2020 ( 48 ) and built the system topology using the CHARMM-36 force field ( 54 ), using the predicted model obtained by the AlphaFold2 algorithm ( 49 ) as the initial protein structure for the simulation. All the details of the performed simulations are available in the SI. 4. Retinal neuron differentiation from human iPSC and treatment. Human iPSCs (SIGi001-A) were differentiated into retinal neurons using a modified multi-step protocol ( 50 ), ( 51 ) detailed in SI. DIV 30 retinal neurons were treated with fibrillated K18 samples for 1, 24, and 48 hours at 37°C and 5% CO 2 . 5. Cytotoxicity analysis. A live-dead assay was conducted with 8 µg/ml FDA for live cells, 20 µg/ml PI for dead cells, and Hoechst for nuclei. Neurons were treated with K18-Heparin and K18-NaTPP (4 and 7 days), stained, and incubated in HEPES-buffered solution. Cell counts were analyzed using ImageJ. Percentages of live and dead cells were calculated as (Live/Total Cells) * 100 and (Dead/Total Cells) * 100, after 24 hours and two weeks. 6. Immunostaining, confocal imaging, and analysis. iPSC-derived retinal neurons were fixed with 4% PFA at day 30, permeabilized with 0.2% Triton X-100, and blocked with 0.1% Tween-20 and 5% goat serum. Cells were incubated overnight with antibodies: TUJ1, pTAU Ser202/Ser205, HT7, p62/SQSTM1, MAP2, and Cleaved caspase 3, followed by AlexaFluor secondary antibodies and Hoechst. Confocal images (2048 x 2048) were acquired using an Olympus iX73 microscope. Cytoskeleton integrity was measured by % area covered by TUJ1 and MAP2, with TUJ1 analysis including binarization and segmentation using ImageJ. Segment length was measured, and % area covered by HT7 quantified total tau. Phospho-Tau Ser202/Ser 205 was measured using "find maxima." P62/SQSTM1 was analyzed as puncta, and its colocalization with rhodamine-K18 was assessed using Mander’s overlap coefficient. Cleaved-caspase 3 positive nuclei were manually counted and calculated as (cleaved-caspase 3 positive cells/total cells) * 100. 7. Statistics. The analysis was conducted on at least three biological replicates per condition. Data were reported as mean ± SEM and plotted using GraphPad Prism 8.0. Statistical analysis involved parametric or non-parametric ANOVA, with treatment as the independent variable. Significant differences were further analyzed using Dunnett, Sidak, or Tukey multiple comparison tests. Significance levels were *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. RESULTS Heparin- and NaTPP-induced K18 fibrils show different fibrillation kinetics and morphology. To evaluate the effects of the two different aggregation agents, heparin and NaTPP, the fibrillated samples were analyzed using a recently developed probe with high affinity for tau-based β-sheet structures, BT1-loaded ferritin (HumAfFt-BT1) ( 45 ), ( 46 ). Different aliquots of fibrils were taken from each sample at various time points and analyzed by HumAfFt-BT1 fluorescence assay (Fig. 1 C). In our experiment, the initial lag phase for both K18-Hep and K18-NaTPP corresponded to 3 days of incubation at 37°C ( 29 ). Over this period, the samples showed a negligible increase in fluorescence due to the formation of numerous nuclei (Fig. 1 C). However, after 4 days, both samples reached the growth phase corresponding to the elongation of the nuclei into oligomers and protofibrils. The fluorescence intensity increased similarly for both K18-Hep and K18-NaTPP. The elongation of the fibrils continued up to the seventh day, in which a significant difference in fluorescence between the samples was observed (Fig. 1 C; K18-Hep-7d VS K18-NaTPP-7d p = 0.0182). K18-NaTPP showed a higher fluorescence intensity, almost double the one of K18-Hep. After 10 days, the growing phase ended, and the fluorescence intensity remained constant, showing a slight decrease after 14 days, probably caused by the formation of very insoluble aggregates (Fig. 1 C). The major differences in fibrillation kinetics were observed during the growth phase, between 4 and 7 days. Therefore, to explore the effects of the NaTPP on the formation and morphology of K18 fibrils, these two time points were further investigated by scanning transmission electron microscopy (STEM). K18 exposed to heparin (1:1 in molar concentrations) showed great difference after 4 and 7 days (Fig. 1 D-F). Population distribution of the K18-Hep sample after 4 days at 37°C resulted in a scarce amount of fibrils (1.6 ± 0.13%), while after 7 days the population of fibrils was richer (19.3 ± 4.25%) (Fig. 1 E; K18-Hep-4d VS K18-Hep-7d p < 0.0001). The differences between these two samples concerned also the length of the fibrils. In fact, after 4 days of exposure to heparin, K18 presented higher fibril variability, whereas the protein showed multiple aggregates with a more consistent and final fibril length after 7 days (Fig. 1 F; K18-Hep-4d VS K18-Hep-7d p = 0.0093). This behavior was similar for K18-NaTPP samples. On average, these fibrils were longer than K18-Hep fibrils (K18-NaTPP > 800 nm, K18-Hep < 400 nm, Fig. 1 F). However, after 4 days at 37°C, the fibrils still showed oscillations in dimension, whilst after 7 days, the length variability was lower, although still present (Fig. 1 F; K18-NaTPP-4d VS K18-NaTPP-7d p=?). The population distribution of K18-NaTPP fibrils after 4 days (2.94 ± 0.72%) was similar to the same sample after 7 days (3.28 ± 1.25%) (Fig. 1 E). These data indicate that K18 exposed to NaTPP produces longer fibrils than heparin, showing fibril maturity after 7 days. Molecular Dynamics Simulations Reveal NaTPP-Induced Aggregation Mechanisms of K18 Tau Protein With the aim of investigating the equilibrium conformational exploration of K18, we performed a one-microsecond molecular dynamics simulation of K18 in the presence of NaTPP. As control we also performed two additional one-microsecond molecular dynamics simulations, considering K18 protein alone in water, and K18 in the presence of sodium monophosphate (Na 2 HPO 4 )(NaP). A set of preliminary analyses (Root Mean Square Deviation (RMSD), radius of gyration, and contact analysis, Fig. 2 A-B-C) for all three simulated systems highlighted that the K18 protein adopts a more compact conformation, regardless of the presence of ligands (see Supplementary Material for more details). To investigate the dynamic-structural stability of the solvent-exposed regions involved in molecular binding, in this specific case, we leverage the experimental knowledge of the system ( 7 ). Therefore, we selected the three interacting glycines (GGG patch: residues 333, 334, and 335) from PDB 5O3L, which are central residues of the interface. A cartoon representation of this structure is shown in Fig. 2 E (left), where the three glycines in question are highlighted in red. We calculated the area of molecular iso-electron density surfaces formed by the three glycines during the molecular dynamics simulation for the three molecular systems separately, with the idea that the larger the area of the considered surface portion, the greater the possibility of interaction. For the two systems that did not form aggregates (Fig. 2 D, free K18, top trace, and K18 with NaP, middle trace), the solvent-exposed area of residues involved in the fibril interface fluctuated significantly throughout the simulation, transitioning from high exposure values (in black), potentially favorable to binding, to low solvent exposure values (in red), indicative of conformations that theoretically hinder binding. Interestingly, when K18 interacted with NaTPP, after approximately 500 ns, it began to explore conformations that did not present a marked structural stability compared to the first part of the simulation (see RMSD and radius of gyration analysis) but maintained a highly stable area value in the interface region. The evidence of this signal in Fig. 2 D (bottom trace) is of great interest, with an average area value of 121Ų, with a standard deviation of 11 Ų. In contrast, during the first half of the simulation, the same system exhibited a more pronounced variability in surface area, with an average of 113 Ų and a standard deviation of 24 Ų. A structural visualization of the surface areas related to the three glycines is shown in Fig. 2 E, where we show a snapshot of the trajectory in which the three glycines were partially buried (Fig. 2 E, middle) and one, belonging to the second half of the simulation of K18 in the presence of NaTPP, in which the three glycines are markedly exposed (Fig. 2 E, right). The stabilization of the solvent-exposed area of ​​residues involved in the fibril interface suggests the importance of stabilizing not only the entire protein structure (probably a necessary condition) but also the binding region (a necessary and sufficient condition). Additionally, based on the ligand-protein distance, we defined the residues in contact with the NaTPP at each frame of the simulation to calculate the relative contact frequency of each residue separately for the first part and the second part of the molecular dynamics simulation (Fig. 2 F, left). In general, the contacts of the K18 residues interacting with NaTPP are largely conserved. However, in Fig. 2 F, right, we highlighted in green the residues that have significantly increased the frequency of contact with the ligand in the second half of the simulation, compared to the first part of the trajectory (55I, 56K, 65Q, 66I, and 81G). On the contrary, in yellow, we reported the residues with many contacts in the first part and a loss of contacts in the second half of the trajectory. The initial residues in the N-terminus of K18 also became consistently interacting in the second half of the simulation, indicating that the new rearrangement of the protein configuration could also be induced by a new interaction between the ligand and the N-terminal of K18. During the first half of the simulation, when the area of the GGG patch involved in the fibril interface was variable and generally less exposed, this region frequently interacted with two sets of residues, one centered around 55I and 56K residues, and the other centered around 65Q and 66I residues. At the end of the first half of the simulation, a significant conformational change occurred in K18, as shown in the previous analysis. In the second half of the simulation, the pairs of residues 55I-56K and 65Q-66I, which formed the basis of a loop created in K18, came closer together and interacted with the ligand (thus moving away from the three glycines belonging to the fibril interface). During the first part of the simulation, these residues were not at the base of the loop and were less constrained. On the contrary, the new configuration restricted the length of this region free to move, making it insufficient to prevent the three glycines from interacting with the solvent. In Fig. 2 F, snapshots of the simulation are shown, depicting the contact between residues 55I, K56, 65Q, and 66I (in green) and the three glycines (in red) during the first phase and their involvement in binding with the NaTPP during the second half of the simulation. This conformation remained locally stable and ensured the constant exposure of the GGG patch, as evident from the molecular surface area plot. Short-Term Cellular Responses to K18 Fibrils: Increased Endogenous Tau and Autophagic Activity To study the impact of K18 fibrils on retinal neurons, we treated DIV 30 human iPSC-derived retinal cultures with 5 µM of K18 fibrils for 24 hours after confirming K18 fibril internalization and toxicity (Supplementary Figure S3, Scheme S3D). Increased endogenous tau protein expression was observed with K18-Hep-4d, K18-Hep-7d, and K18-NaTPP-4d treatments but not with K18-NaTPP-7d. This was verified using an anti-total tau (HT7) antibody that recognizes the "PPGQK" epitope of human tau, absent in K18 (Fig. 1 A), thus detecting only endogenous tau (Fig. 3 A- 3 B). Similar results were found with the cytoskeletal marker MAP2 (Supplementary Fig. 4). Interestingly, K18-NaTPP-4d treatment led to the highest expression of endogenous tau protein compared to other samples (Fig. 3 B). This increase in tau was not associated with changes in β-III Tubulin (TUJ1) (Supplementary Fig. S4), supporting the hypothesis that exogenous fibrils trigger a stress response, leading to cellular upregulation of endogenous tau ( 16 ), ( 52 ). We also observed increased p62/SQSTM1 puncta following K18 fibril treatment (Fig. 3 C- 3 D). Notably, p62 puncta were significantly more abundant in cultures treated with K18-NaTPP-4d than those treated with K18-NaTPP-7d or K18-Hep-4d/7d (Fig. 3 D). Additionally, p62 puncta colocalized with exogenous K18 fibrils, with the strongest colocalization observed in K18-Hep-4d and K18-NaTPP-4d treatments (Fig. 3 E- 3 F). These results indicate an increase in autophagic flux after 24 hours, aligning with p62 neuroprotective role in reducing oligomeric tau. However, we did not observe Cleaved-caspase3 activation, indicating that short-term K18 exposure does not induce apoptosis (Fig. 3 G-H). Long-Term Neurodegenerative Effects of K18 Fibrils: Cytoskeleton Alteration and Apoptotic Pathway Activation We assessed the impact of K18 fibril treatment on neuronal dysfunction and neurodegeneration two weeks after incubation (Fig. 4 A). A live-dead assay revealed a notable reduction in live cells, with a 40% decrease in cultures internalizing K18-Hep-4d and K18-Hep-7d fibrils, a 45% decrease after K18-NaTPP-4d treatment, and a 30% decrease following K18-NaTPP-7d exposure (Fig. 4 B). Immunostaining showed a significant decrease in the neurite marker TUJ1 area in cultures treated with K18-Hep-7d, K18-NaTPP-4d, and K18-NaTPP-7d fibrils compared to controls (Fig. 4 C- 4 D left). Additionally, fibril-treated cultures exhibited smaller TUJ1 fragments (Fig. 4 D middle and right; Supplementary Fig. S5). These findings indicate that internalized fibrils gradually disrupt the neuronal cytoskeleton, with more severe effects observed in K18-NaTPP fibril-treated cultures. In this model, fibril treatment also led to the formation of hyperphosphorylated pathological tau ( 16 ). We measured tau phosphorylation at the Ser-202, Thr-205 (AT8) epitopes (Fig. 4 C) and found a significant increase in phosphorylated tau within neurites of treated cells compared to untreated ones (Fig. 4 C, zoomed panel; Fig. 4 E). We then explored the role of p62/SQSTM1 in processing phosphorylated tau. Immunofluorescence analysis revealed a marked increase in p62/SQSTM1 puncta in cultures treated with K18-Hep fibrils, but not in those treated with K18-NaTPP fibrils (Fig. 4 F- 4 G). Additionally, we observed a significant activation of the apoptotic pathway two weeks post-treatment, indicated by an increased Cleaved Caspase-3 signal (Fig. 4 H). Collectively, these findings suggest that K18 fibril treatment induces long-term neurite degeneration and neuronal loss, which contribute to neurodegeneration and ultimately lead to cell death. DISCUSSION In this study, we report the differential effects of K18 tau fibrils, induced by heparin and NaTPP, on human iPSC-derived retinal neurons ( 50 , 51 ) over short-term and long-term exposures. NaTPP and heparin impact fibrillation kinetics ( 29 ) and morphology differently, leading to distinct structural and cellular responses. Notably, NaTPP-induced fibrils showed a more rapid and extensive aggregation process compared to heparin, and produced longer, more stable fibrils over seven days. These distinctions in fibrillation dynamics translate into varied cellular effects, shedding light on the mechanisms by which different fibril structures interact with neuronal cells and contribute to neurodegeneration. Short-Term Cellular Responses: Tau Induction and Autophagy Activation Our findings suggest that K18 fibrils induce an acute stress response in retinal neurons, leading to increased expression of endogenous tau protein. This effect was most pronounced in cultures treated with K18-NaTPP-4d, which showed the highest tau upregulation among the fibril types. The selective increase in tau, confirmed via anti-tau (HT7) immunostaining, implies that exogenous fibrils may stimulate a compensatory cellular mechanism aimed at buffering against tau aggregation stress. Furthermore, K18-NaTPP-4d also caused a notable rise in autophagic flux, as indicated by the substantial increase in p62/SQSTM1 puncta, colocalized with exogenous K18 fibrils, suggesting that autophagy serves a neuroprotective role by targeting tau aggregates for degradation. Importantly, there was no evidence of Cleaved Caspase-3 activation during this short-term exposure, indicating that while K18 fibrils impose cellular stress, they do not immediately trigger apoptosis within 24 hours. Long-Term Neurodegenerative Effects: Cytoskeleton Disruption and Apoptosis Prolonged exposure to K18 fibrils led to marked neurodegenerative outcomes, highlighting the cumulative impact of sustained fibril presence on neuronal integrity. Two weeks post-treatment, we observed a significant reduction in cell viability, with the most severe decreases in cultures treated with K18-Hep-4d, K18-Hep-7d, and K18-NaTPP-4d fibrils. This decline in live cells, accompanied by a reduction in TUJ1-labeled neurite area, indicates that prolonged fibril internalization progressively disrupts the neuronal cytoskeleton. Furthermore, fibril-treated cells exhibited hyperphosphorylated tau at the AT8 epitope, a hallmark of tauopathy, suggesting that continuous fibril exposure fosters pathogenic tau modifications that contribute to structural degeneration. Interestingly, long-term exposure also differentially impacted p62/SQSTM1 activity across fibril types. While K18-Hep fibrils prompted an increase in p62/SQSTM1 puncta, this was not observed with K18-NaTPP fibrils. This suggests that K18-Hep fibrils may induce a stronger autophagic response to manage phosphorylated tau, whereas K18-NaTPP fibrils may engage alternative pathways or mechanisms. The eventual activation of the apoptotic pathway, as evidenced by Cleaved Caspase-3 staining, underscores a shift from protective responses to neurodegenerative processes, marking a tipping point where cellular defenses become insufficient to counteract fibril-induced toxicity. Molecular Dynamics Insights: Structural Basis of NaTPP-Induced Aggregation The K18-NaTPP-7d fibrils appear to have adopted a helicoidal mature structure, contrasting with the shorter, entangled aggregates observed in K18-Hep fibrils and K18-NaTPP-4d, which may be still in the protofibril phase ( 54 ), ( 55 ), ( 56 ). Over time, however, both NaTPP- and heparin-induced fibrils led to tau pathology and neuronal death. In line with the hypothesis that mature fibrils, rather than intermediates, are less toxic, these differences in fibril behavior may be related to variations in their size and morphology. K18-Hep-4d fibrils were smaller and more heterogeneous, while K18-Hep-7d fibrils formed longer, more numerous structures, though their length did not exceed 400 nm. Conversely, K18-NaTPP-4d fibrils were more homogeneous and over 800 nm long, resembling the morphology of 7-day fibrils. Alongside longer polyphosphates, our results indicated that even short-chain polyphosphates like NaTPP can reduce the lag phase of fibril formation and promote elongation ( 47 , 53 ). Molecular dynamics simulations further confirmed this hypothesis. The root mean square deviation (RMSD) and radius of gyration indicated distinct conformational behaviors of Tau protein in water, phosphate buffer, and NaTPP. In this condition, K18 exhibited rapid compaction early in the simulation, a feature absent in the other systems, which likely indicates an aggregated conformation. A key finding was the behavior of the GGG patch (residues 333–335), crucial for tau fibril formation. In the K18-NaTPP system, the GGG patch remained solvent-exposed, suggesting that NaTPP stabilizes the aggregation-prone conformation of K18. Protein-ligand interactions also played a role, with specific residues (55I, 56K, 65Q, 66I, 81G) forming stable contacts with NaTPP, further stabilizing the aggregation-prone state by maintaining the exposure of the GGG patch. Although the mechanism that induces tau fibril formation is highly complex and likely results from the interaction of multiple concomitant factors, our study highlighted an unexplored role of NaTPP, whose presence is physiologically relevant in neurons ( 27 , 28 , 29 ). The physiopathological relevance of NaTPP-induced tau fibrillation lies in its ability to mimic endogenous polyphosphates (polyP), which significantly influence neuronal function and excitability but also participate in pathological tau aggregation. NaTPP-induced tau fibrillation highlights the dual nature of polyP in neuronal homeostasis: while polyP modulates ion channels, regulates calcium signaling, and offers neuroprotective effects, it also promotes tau aggregation under certain conditions ( 28 , 29 , 57 , 58 ). This aggregation contributes to neurotoxicity by enhancing pathological tau structures associated with neurodegenerative processes, suggesting that while polyP plays a critical role in neuronal health, its impact on tau fibrillation may drive toxic outcomes in disease states. Overall, our study demonstrated that the short-chain NaTPP plays a key role in tau folding into tangles due to molecular interactions with specific residues of K18 and in the development of pathological cellular responses in iPSC cells, providing a basis for further investigation into their potential role in neurodegenerative disease. Abbreviations AD Alzheimer's disease Aβ β-amyloid DIV Day in vitro FDA Fluorescein diacetate FOV Field of view FTD Fronto temporal dementia GGG 3 interacting glycines hiPSC Human induced pluripotent stem cells HumAfFt-BT1 BT1-loaded ferritin iPSC Induced pluripotent stem cells IPTG Isopropyl β-D-1-tiogalattopiranoside K18 Microtubule-binding domain MTBR Microtubule-binding repeat region NaP Sodium monophosphate NaTPP Sodium tripolyphosphate NES HEPES-buffered external solution NFT Neurofibrillary tangles PDF Probability density functions PFA Paraformaldehyde PHF Paired helical filaments PI Propidium Iodide PolyPs Polyphosphates RMSD Root mean square deviation SF Straight filaments STEM Scanning transmission electron microscopy TCEP Tris(2-carboxyethyl) phosphine hydrochloride ThT Thioflavin T Declarations AVAILABILITY OF DATA AND MATERIALS The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request. For the analysis of imaging data, we utilized custom code developed in MathWorks Matlab (version 2016b), which is available upon request. All the protein structures used in this paper are available in the Protein Data Bank. The Data generated with molecular dynamics and molecular docking will be available upon request. ACKNOWLEDGEMENTS The authors wish to thank the Center for Life Nano and Neuroscience Imaging Facility, Istituto Italiano di Tecnologia. FUNDING This work was supported by MUR PRIN 2022 (CUP: 2022CFP7RF, to SDA and PB, EM). This work was supported by Regione LAZIO Lazio-Innova; POR FESR Lazio 2014-2020 (A0375-2020-36549, CUP: B85F20003340002 to PB). This research was also funded by the D-Tails-IIT Joint Lab (to SDA, YG, AB), the Regione Lazio FSE 2014–2020 (19036AP000000019 and A0112E0073) grants (to SDA), Sapienza University grants (RM118163E0297F84, PH12017270934C3C, MA32117A7B698029 and RM1231889BB77735to SDA; RM123188F763A9D5 to PB), and Fondazione Istituto Italiano di Tecnologia (to LM). LM was also supported by the PhD program in Life Science at Sapienza University in Rome. SDA was also supported by Progetto ECS 0000024 Rome Technopole, - CUP B83C22002820006, PNRR Missione 4 Componente 2 Investimento 1.5, finanziato dall’Unione europea – NextGenerationEU. SDA was also supported by the Italian Ministry of Health - Alternative Methods to Animal Testing Grant 2023 (NEURO-3R). AB, GR and PB were also supported by Project "National Center for Gene Therapy and Drugs based on RNA Technology" (CN00000041) financed by NextGenerationEU PNRR MUR—M4C2—Action 1.4-Call "Potenziamento strutture di ricerca e creazione di "campioni nazionali di R&S" (CUP J33C22001130001). LB was supported by Progetto Avvio alla Ricerca 2022 (University “La Sapienza,” Rome, Italy). The research leading to these results was also supported by the European Research Council through its Synergy grant program, project ASTRA (grant agreement No 855923) and by the European Innovation Council through its Pathfinder Open Programme, project ivBM-4PAP (grant agreement No 101098989). ETHICS DECLARATIONS Ethics approval and consent to participate The use of hiPSC has been approved by the Ethical Committee for Translational Research (CERT) Sapienza University (Aut. n. 5/2022). Consent for publication Not applicable. Competing interests The funders had no role in the study design, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results. YG is employed by D-Tails s.r.l.; SDA is a scientific advisor of D-Tails s.r.l. The remaining authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest. AUTHOR CONTRIBUTIONS LB performed biochemical experiments and related chemical analysis; YG performed retinal differentiation experiments (confocal microscopy, immunofluorescence, internalization) and related analysis; LM performed retinal differentiation experiments and molecular and cellular characterization; SG wrote the MATLAB code and performed quantification of confocal acquisitions; FM performed STEM acquisition; MVF performed spectral analysis; ST and LD performed EM analysis; AB and PB designed the biochemical experimental strategy; SDA designed biological experimental strategy; GR and EM designed the computational approach; EM performed the computational analysis; AB, GR, PB, EM and SDA conceived the study; PB, EM and SDA designed the experiments, interpreted the results, and wrote the manuscript. The manuscript was written through the contributions of all authors. All authors have approved the final version of the manuscript. References Binder LI, Frankfurter A, Rebhun LI. 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Pollack SJ, Trigg J, Khanom T, Biasetti L, Marshall KE, Al-Hilaly YK, et al. Paired Helical Filament-Forming Region of Tau (297–391) Influences Endogenous Tau Protein and Accumulates in Acidic Compartments in Human Neuronal Cells. Journal of Molecular Biology. 2020;432(17):4891–907. Jorgensen WL, Chandrasekhar J, Madura JD, Impey RW, Klein ML. Comparison of simple potential functions for simulating liquid water. The Journal of Chemical Physics. 1983;79(2):926–35. Cremers CM, Knoefler D, Gates S, Martin N, Dahl JU, Lempart J, et al. Polyphosphate: A Conserved Modifier of Amyloidogenic Processes. Molecular Cell. 2016;63(5):768–80. Murphy RM. Kinetics of amyloid formation and membrane interaction with amyloidogenic proteins. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2007;1768(8):1923–34. Kumar AV, Mills J, Lapierre LR. Selective Autophagy Receptor p62/SQSTM1, a Pivotal Player in Stress and Aging. Front Cell Dev Biol. 2022;10:793328. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5409787","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":378672582,"identity":"60c8d43f-7c14-4f35-bd60-b84e3755b080","order_by":0,"name":"Paola 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12:11:21","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5409787/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5409787/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41419-025-07662-5","type":"published","date":"2025-05-09T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":70311804,"identity":"19316fae-b1d7-4d56-a295-80b94a8ce502","added_by":"auto","created_at":"2024-12-02 04:37:52","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1193432,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKinetics of aggregation and structural characterization over time of K18 domain with heparin and NaTPP cofactors.\u003cbr\u003e\nA) \u003c/strong\u003eSchematic view of the full-length form of protein tau (0N4R). Recombinant K18 domain from 244-372 and the mutation C249S are shown. HT7 recognition site is also indicated in green; \u003cstrong\u003eB)\u003c/strong\u003e Chemical structures of anionic compounds used in the same ratio 1:1 = protein:cofactor; \u003cstrong\u003eC)\u003c/strong\u003e Representative plot showing the fibril formation using BT1 fluorescence as a function of time in the presence of non-fibrillated K18, 100 µM, and fibrillated K18-Hep, 100 µM (orange curve) and K18-NaTPP 100 µM (blue curve) at 37°C at different time points. All spectra were obtained using a λex = 530 nm, λem = 565 nm. All data are mean ± SEM, n=3, two-way ANOVA and Sidak's multiple comparisons post hoc test *p\u0026lt;0.05; \u003cstrong\u003eD and D’)\u003c/strong\u003e Representative STEM images of negative stained K18 fibrils, at lower (D) and higher (D’) magnification (scale bars 1 μm and 200 nm, respectively). The fibrils were formed with heparin after 4 days (i) and 7 days (ii), and with NaTPP after 4 days (iii) and 7 days (iv); \u003cstrong\u003eE) \u003c/strong\u003emeasurement of population distribution of fibrils in all four samples calculated from STEM images (All data are mean ± SEM, n=FOV=3, one-way ANOVA and Tukey's multiple comparisons post hoc test****p\u0026lt;0.0001); \u003cstrong\u003eF)\u003c/strong\u003e measurement of average length of K18 fibrils in all four samples calculated from STEM images (All data are mean ± SEM, n=50, Kruskal Wallis and Dunn’s multiple comparisons post hoc test **p\u0026lt;0.01, ****p\u0026lt;0.0001).\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-5409787/v1/c490a549cc474f9ce290f66c.png"},{"id":70311802,"identity":"fd8abc09-4676-4602-a67d-dffa0e2e89c5","added_by":"auto","created_at":"2024-12-02 04:37:51","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":444799,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMolecular dynamics simulations analysis.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003eFor each of the three simulated systems (K18 in water in gray, K18 with NaP, in green, and K18 with NaTPP, in orange), the RMSD values as a function of time are reported. Additionally, the probability density functions (PDF) of the RMSD values related to the three systems are shown in the inset. \u003cstrong\u003eB)\u003c/strong\u003e Two cartoon representations of two snapshots from the K18 with NaTPP simulation are shown, one with high (less compact form) and one with low radius of gyration (more compact form). \u003cstrong\u003eC)\u003c/strong\u003e Radius of gyration values as a function of time with the corresponding probability density functions in the inset. \u003cstrong\u003eD)\u003c/strong\u003e For each simulated system, we report the surface area of the molecular surface patch formed by the three consecutive glycine residues directly involved in the fibril interface, whose structure is experimentally known. The surface area value, calculated by summing the contributions of the selected residues' areas, is shown for each frame of the three molecular dynamics simulations. In black are the surface area values greater than 100 Ų (more exposed residues) and in red are the surface area values less than 100 Ų (less exposed residues). \u003cstrong\u003eE)\u003c/strong\u003e On the left is a cartoon representation of the TAU fibril (PDB code 5O3L), with a focus on a single layer of the fibril itself, where the solvent-accessible area of the two sets of three interacting glycines is shown in red. On the right are two cartoon representations of different simulation snapshots: one where the area of the three glycines is below the threshold value of Ų, and one where the area is above the threshold, showing greater exposure. \u003cstrong\u003eF)\u003c/strong\u003e On the left are the relative contact frequencies between each residue of the K18 protein and the monophosphate molecule. The top barplot corresponds to the first half of the simulation, and the bottom barplot corresponds to the second half of the simulation. On the right are snapshots of the K18 with NaTPP simulation: two snapshots from the first half of the simulation (where the residues shown in green interact with the three glycines involved in the fibril interface) and two snapshots from the second half of the simulation (where the same residues shown in green interact with the ligand). In yellow are the residues interacting with the ligand only in the first part of the simulation.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-5409787/v1/a202c3b497c74dfd80062a40.png"},{"id":70311803,"identity":"9ab14ec0-a11c-46af-86c6-5a55082f0506","added_by":"auto","created_at":"2024-12-02 04:37:52","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1750705,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eShort-term effects of tau fibrils on iPSC-derived retinal neurons.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003eImmunostaining for total tau HT7 (magenta) and neurite marker β-III-tubulin TUJ1 (green). Nuclei were stained with HOECHST (blue). Scale bar 25 µm; \u003cstrong\u003eB)\u003c/strong\u003eBar graph represents the % of area covered by total tau in retinal neurons 24 hours after K18-tau seeding (n=FOV=15, one way ANOVA and Tukey’s multiple comparisons post hoc test: K18-Hep-4d VS Untreated p=0.0545, K18-Hep-7d VS Untreated p=0.0040, K18-NaTPP-4d VS Untreated p\u0026lt;0.0001, K18-NaTPP-4d VS K18-Hep-4d p=0.0073, K18-NaTPP-4d VS K18-NaTPP-7d p=0.0002); \u003cstrong\u003eC)\u003c/strong\u003e Representative confocal images of retinal neurons immunostained with MAP2 (gray), p62/SQSTM1 (yellow) and HOECHST to stain nuclei (blue). Scale bar 50 µm. Zoomed images show p62 puncta within MAP2 positive neurite; \u003cstrong\u003eD)\u003c/strong\u003eQuantification of p62/SQSTM1 puncta. (n=FOV=15, one-way ANOVA and Tukey’s multiple comparisons post hoc test: K18-NaTPP-4d VS Untreated p=0.0074, K18-NaTPP-4d VS K18-NaTPP-7d p=0.0162); \u003cstrong\u003eE)\u003c/strong\u003e K18-seed was conjugated with rhodamine (red) and p62/SQSTM1 puncta (yellow) that co-localize with K18 fibrils are highlighted with arrow; Scale bar 10 µm; \u003cstrong\u003eF)\u003c/strong\u003e Bar charts showing the Mander’s colocalization coefficient of k18-fibrils and p62/SQSTM1 in untreated and treated retinal neurons. (n=FOV=6, Kruskal Wallis and Dunn’s multiple comparisons post hoc test: K18-Hep-4d VS Untreated p=0.0177, K18-NaTPP-4d VS Untreated p=0.0493); \u003cstrong\u003eG)\u003c/strong\u003eRepresentative confocal images of iPSC-derived retinal neurons stained with MAP2 (gray), CLEAVED-CASPASE 3 (red) and HOECHST (blue). Scale bar 50 µm; \u003cstrong\u003eH)\u003c/strong\u003e Bar graph represents the percentage of cleaved-caspase 3 positive nuclei normalized to the total number of nuclei. (n=FOV=15, Kruskal-Wallis test, and Dunn’s multiple comparison post hoc test).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-5409787/v1/9758ae5a1e533dbc5e90c50f.png"},{"id":70311801,"identity":"1297f130-7777-42c9-8d34-733cacb3f831","added_by":"auto","created_at":"2024-12-02 04:37:51","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1590728,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eLong-term effects of tau fibrils on iPSC-derived retinal neurons.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA)\u003c/strong\u003e Schematic representation of the experimental plan used to evaluate the long-term effect of K18 fibrils in hiPSC-derived retinal neurons. DIV 30 retinal neurons were treated with K18-Hep-4d, K18-NaTPP-4d, K18-Hep-7d, and K18-NaTPP-7d for 24 hours. Two weeks after treatment confocal microscopy experiments were performed in live-imaging or after fixation Created with \u003ca href=\"http://biorender.com/\"\u003eBiorender.com\u003c/a\u003e; \u003cstrong\u003eB)\u003c/strong\u003e Bar charts show the effect of different K18 fibrils on cell survival. Significant differences are reported compared to the positive control (n=FOV=9, One-way ANOVA and Tukey’s multiple comparisons post hoc test: K18-Hep-4d VS Untreated p\u0026lt;0.0001, K18-Hep-7d VS Untreated p\u0026lt;0.0001, K18-NaTPP-4d VS Untreated p\u0026lt;0.0001, K18-NaTPP-7d VS Untreated p\u0026lt;0.0001); \u003cstrong\u003eC)\u003c/strong\u003e Immunostaining for neurite marker β-III-tubulin TUJ1 (green) and phospho-tau (Ser202, Thr205) (AT8, magenta) two weeks after exposure to K18 fibrils. Scale bar 20 \u0026nbsp;µm; \u003cstrong\u003eD)\u003c/strong\u003eBar graphs represent the % of area covered by TUJ1 (left panel, n=FOV=15, One-way ANOVA and Tukey’s multiple comparisons post hoc test: K18-Hep-7d VS Untreated p=0.0059, K18-NaTPP-4d VS Untreated p=0.0074, K18-NaTPP-7d VS Untreated p=0.0306), the number (middle panel, n=FOV=15, Kruskal Wallis and Dunn’s multiple comparisons post hoc test: K18-Hep-4d VS Untreated p=0.0208, K18-Hep-7d VS Untreated p=0.0025, K18-NaTPP-4d VS Untreated p=0.0149, K18-NaTPP-7d VS Untreated p=0.0002); and the length (right panel, n= number of segments, Kruskal Wallis and Dunn’s multiple comparisons post hoc test: K18-Hep-4d VS Untreated p=0.0451, K18-Hep-7d VS Untreated p\u0026lt;0.0001, K18-NaTPP-4d VS Untreated p\u0026lt;0.0001, K18-NaTPP-7d VS Untreated p=0.0003) of TUJ1 segments in retinal neurons; \u003cstrong\u003eE)\u003c/strong\u003e Bar graph represent the puncta number of AT8 (p-tau Ser202-Thr205) (n=FOV=15, one-way ANOVA and Sidak’s multiple comparisons post hoc test: K18-Hep-4d VS Untreated p=0.0002, K18-Hep-7d VS Untreated p=0.0138, K18-NaTPP-4d VS Untreated p=0.0242, K18-NaTPP-7d VS Untreated p=0.0046); \u003cstrong\u003eF)\u003c/strong\u003e Representative confocal images of retinal neurons immunostained with p62/SQSTM1 (yellow) and neurite marker MAP2 (gray). Nuclei were stained with HOECHST (blue). Scale bar 50 µm. \u0026nbsp;Zoomed images show p62 puncta within MAP2 positive neurite; \u003cstrong\u003eG)\u003c/strong\u003eQuantification of p62/SQSTM1 puncta (n=FOV=15, Kruskal Wallis and Dunn’s multiple comparisons post hoc test: K18-Hep-4d VS Untreated p=0.0033, K18-Hep-7d VS Untreated p=0.0019); \u003cstrong\u003eH) \u003c/strong\u003eRepresentative confocal images of iPSC-derived retinal neurons stained with MAP2 (gray), CLEAVED-CASPASE 3 (red) and HOECHST (blue). Scale bar 50 µm; \u003cstrong\u003eI)\u003c/strong\u003e Bar graph represents the percentage of cleaved-caspase 3 positive nuclei in retinal neurons, normalized to the total number of nuclei. Significant differences are reported compared to the control (n=FOV=15, One-way ANOVA and Tukey’s multiple comparisons post hoc test: K18-Hep-4d VS Untreated p=0.0174, K18-Hep-7d VS Untreated p=0.0271, K18-NaTPP-4d VS Untreated p=0.0115, K18-NaTPP-7d VS Untreated p= 0.0242).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-5409787/v1/f0e3c7bd462fa2ea2af569b5.png"},{"id":82412371,"identity":"8fed53ae-66b4-4969-9470-0ff5f95fb09d","added_by":"auto","created_at":"2025-05-10 07:08:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":6087600,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5409787/v1/e685e45b-14da-42fb-8ca1-109e678904d7.pdf"},{"id":70311805,"identity":"d01b8f8e-feb3-4869-a8d4-99c0f5c922b1","added_by":"auto","created_at":"2024-12-02 04:37:52","extension":"docx","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":4860627,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryInformationBaroloetal.CellDeathandDisease.docx","url":"https://assets-eu.researchsquare.com/files/rs-5409787/v1/bc518570bab588c54db442a1.docx"}],"financialInterests":"(Not answered)","formattedTitle":"Short-Chain Polyphosphates Induce Tau Fibrillation and Neurotoxicity in Human iPSC-Derived Retinal Neurons","fulltext":[{"header":"INTRODUCTION","content":"\u003cp\u003eTau is a microtubule-associated protein primarily found in neurons, playing a critical role in maintaining axonal structural integrity (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e),(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Its dysregulation leads to neurofibrillary tangles (NFTs), protein aggregates characteristic of neurodegenerative diseases such as Alzheimer\u0026rsquo;s disease (AD) and frontotemporal dementia (FTD) (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), (4), (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e5\u003c/span\u003e). In pathological conditions, tau undergoes conformational changes, transitioning from monomers to oligomers and eventually mature filaments (\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e6\u003c/span\u003e), (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The neurotoxicity is primarily associated with tau oligomers, which accumulate intracellularly during fibrils formation and spread between neurons, contributing to disease progression (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e8\u003c/span\u003e), (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e9\u003c/span\u003e), (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e10\u003c/span\u003e), (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e11\u003c/span\u003e). Recent studies have demonstrated that tau oligomers generated \u003cem\u003ein vitro\u003c/em\u003e can be internalized by human neurons derived from induced pluripotent stem cells (iPSCs) and murine models (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e12\u003c/span\u003e), (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e13\u003c/span\u003e), (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Once internalized, these oligomers act as templates, seeding the aggregation of endogenous tau and triggering a cascade that accelerates fibril formation, thereby enabling the long-term observation of neurodegenerative processes (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e15\u003c/span\u003e), (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e16\u003c/span\u003e), (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e17\u003c/span\u003e), (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e18\u003c/span\u003e). To model these aggregation dynamics, the microtubule-binding repeat region (MTBR), or K18 domain (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e19\u003c/span\u003e),(\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e20\u003c/span\u003e),(\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e21\u003c/span\u003e) is typically incubated with polyanionic compounds like polyunsaturated fatty acids, RNA, and polyglutamate (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e), (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e22\u003c/span\u003e), (\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e23\u003c/span\u003e), (\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e24\u003c/span\u003e), (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Heparin is frequently used for this purpose, but its use presents significant limitations, as it does not participate in tau fibrillation in vivo, and its polymorphic nature complicates the study of tau behavior under physiological conditions (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e22\u003c/span\u003e), (\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e26\u003c/span\u003e). Polyphosphates (polyPs) have emerged as promising physiologically relevant alternatives for inducing tau fibrillation. Present in the extracellular space of the brain, polyPs play roles in cellular energy homeostasis, inflammation, and cell signaling (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e27\u003c/span\u003e),(\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e28\u003c/span\u003e), (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e29\u003c/span\u003e), (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e30\u003c/span\u003e), (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e31\u003c/span\u003e), (\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e32\u003c/span\u003e),(\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e33\u003c/span\u003e),(\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e34\u003c/span\u003e)(\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e35\u003c/span\u003e). More importantly, polyPs compete with tubulin for binding tau, promoting its aggregation into amyloid fibrils and potentially contributing to AD pathology (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e28\u003c/span\u003e), (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e29\u003c/span\u003e), (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e35\u003c/span\u003e). Tau fibrils formed with polyPs exhibit reduced cytotoxicity, especially in the early stages of aggregation, likely due to the reduced presence of toxic oligomers and protofibrils (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e36\u003c/span\u003e), (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e37\u003c/span\u003e), (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e38\u003c/span\u003e),(\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e39\u003c/span\u003e). Here, we report for the first time the possibility of obtaining mature fibrils of the K18 domain through a naturally occurring cofactor sodium tripolyphosphate (NaTPP) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA-B). Our findings revealed a fundamental difference in aggregation kinetics and the number and length of filaments compared to heparin, enlightening a significant difference in the transition from smaller oligomeric species to longer-growing fibrils. Molecular dynamics simulations of K18 enlighten the exposure of three residues most involved in the fibril interface (GGG). The computational study shows clear compaction of K18 that, only in the presence of NaTPP, does an ensemble of conformations with a clear solvent exposure of the region involved in the tau fibril interface. A detailed analysis of protein-ligand contacts highlighted a set of key K18 residues, whose intermolecular contacts with NaTPP would ensure a stable area accessible to the solvent of the GGG patch, making the K18 conformation prone to binding with itself. The role of K18-NaTPP-induced aggregates was studied \u003cem\u003ein vitro\u003c/em\u003e using human iPSC differentiated into retinal neurons. K18-NaTPP fibrillar structures, at lower incubation time, are generally reported as less cytotoxic than heparin, triggering a cellular protective response in the short term. However, this condition dramatically changed over a longer period, leading to the activation of the apoptotic pathway. Based on these findings, short polyphosphates may act as a potential catalyst for internalizing preformed K18-fibrils into iPSC retinal neurons, promoting tau-related conditions compatible with a pathological environment, including neurodegenerative diseases like AD and FTD.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e1. Tau protein construct design, expression and purification\u003c/h2\u003e \u003cp\u003eThis study used the K18 domain referred to as the MTBR domain of tau isoform 0N4R (residues 244 to 372) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA) containing a point mutation C291S (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e40\u003c/span\u003e), (\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e41\u003c/span\u003e), (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Protein production and purification are described in SI (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e43\u003c/span\u003e), (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e44\u003c/span\u003e). The conformational assembly of K18 was analyzed by size exclusion chromatography using a HiLoad\u0026trade; 26/600 Superdex 75 (Cytiva).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003e2. Formation and Fluorescence analysis of K18 fibrils\u003c/h3\u003e\n\u003cp\u003eK18 100 \u0026micro;M in PBS was reduced using 1 mM TCEP 10 minutes at 55\u0026deg;C. Sodium heparin or NaTPP was added in a 1:1 ratio to initiate tau protein aggregation \u003cem\u003ein vitro\u003c/em\u003e. The samples (K18-Hep and K18-NaTPP) were kept at 37\u0026deg;C under rotation at 150 rpm for several time points. The fibrillation kinetics was analyzed using the BODIPY-based probe BT1, loaded into humanized ferritin nanocages due to the limited specificity of thioflavin T (ThT) towards K18 fibrils (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e45\u003c/span\u003e), (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e46\u003c/span\u003e), (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e47\u003c/span\u003e). All the measurements were made by using the RF-6000 fluorimeter (Shimadzu RF-6000). Further details and the preparation of K18 fibrils for STEM imaging are given in SI.\u003c/p\u003e\n\u003ch3\u003e3. Computational Methods\u003c/h3\u003e\n\u003cp\u003eFor each of the molecular systems considered in this work, we used Gromacs 2020 (\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e48\u003c/span\u003e) and built the system topology using the CHARMM-36 force field (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e54\u003c/span\u003e), using the predicted model obtained by the AlphaFold2 algorithm (\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e49\u003c/span\u003e) as the initial protein structure for the simulation. All the details of the performed simulations are available in the SI.\u003c/p\u003e \u003cp\u003e \u003cb\u003e4. Retinal neuron differentiation from human iPSC and treatment.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eHuman iPSCs (SIGi001-A) were differentiated into retinal neurons using a modified multi-step protocol (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e50\u003c/span\u003e), (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e51\u003c/span\u003e) detailed in SI. DIV 30 retinal neurons were treated with fibrillated K18 samples for 1, 24, and 48 hours at 37\u0026deg;C and 5% CO\u003csub\u003e2\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003e \u003cb\u003e5. Cytotoxicity analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eA live-dead assay was conducted with 8 \u0026micro;g/ml FDA for live cells, 20 \u0026micro;g/ml PI for dead cells, and Hoechst for nuclei. Neurons were treated with K18-Heparin and K18-NaTPP (4 and 7 days), stained, and incubated in HEPES-buffered solution. Cell counts were analyzed using ImageJ. Percentages of live and dead cells were calculated as (Live/Total Cells) * 100 and (Dead/Total Cells) * 100, after 24 hours and two weeks.\u003c/p\u003e \u003cp\u003e \u003cb\u003e6. Immunostaining, confocal imaging, and analysis.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eiPSC-derived retinal neurons were fixed with 4% PFA at day 30, permeabilized with 0.2% Triton X-100, and blocked with 0.1% Tween-20 and 5% goat serum. Cells were incubated overnight with antibodies: TUJ1, pTAU Ser202/Ser205, HT7, p62/SQSTM1, MAP2, and Cleaved caspase 3, followed by AlexaFluor secondary antibodies and Hoechst. Confocal images (2048 x 2048) were acquired using an Olympus iX73 microscope. Cytoskeleton integrity was measured by % area covered by TUJ1 and MAP2, with TUJ1 analysis including binarization and segmentation using ImageJ. Segment length was measured, and % area covered by HT7 quantified total tau. Phospho-Tau Ser202/Ser 205 was measured using \"find maxima.\" P62/SQSTM1 was analyzed as puncta, and its colocalization with rhodamine-K18 was assessed using Mander\u0026rsquo;s overlap coefficient. Cleaved-caspase 3 positive nuclei were manually counted and calculated as (cleaved-caspase 3 positive cells/total cells) * 100.\u003c/p\u003e \u003cp\u003e \u003cb\u003e7. Statistics.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eThe analysis was conducted on at least three biological replicates per condition. Data were reported as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM and plotted using GraphPad Prism 8.0. Statistical analysis involved parametric or non-parametric ANOVA, with treatment as the independent variable. Significant differences were further analyzed using Dunnett, Sidak, or Tukey multiple comparison tests. Significance levels were *p\u0026thinsp;\u0026le;\u0026thinsp;0.05, **p\u0026thinsp;\u0026le;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026le;\u0026thinsp;0.001, ****p\u0026thinsp;\u0026le;\u0026thinsp;0.0001.\u003c/p\u003e"},{"header":"RESULTS","content":"\u003cp\u003e \u003cb\u003eHeparin- and NaTPP-induced K18 fibrils show different fibrillation kinetics and morphology.\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo evaluate the effects of the two different aggregation agents, heparin and NaTPP, the fibrillated samples were analyzed using a recently developed probe with high affinity for tau-based β-sheet structures, BT1-loaded ferritin (HumAfFt-BT1) (\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e45\u003c/span\u003e), (\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e46\u003c/span\u003e). Different aliquots of fibrils were taken from each sample at various time points and analyzed by HumAfFt-BT1 fluorescence assay (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). In our experiment, the initial lag phase for both K18-Hep and K18-NaTPP corresponded to 3 days of incubation at 37\u0026deg;C (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e29\u003c/span\u003e). Over this period, the samples showed a negligible increase in fluorescence due to the formation of numerous nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). However, after 4 days, both samples reached the growth phase corresponding to the elongation of the nuclei into oligomers and protofibrils. The fluorescence intensity increased similarly for both K18-Hep and K18-NaTPP. The elongation of the fibrils continued up to the seventh day, in which a significant difference in fluorescence between the samples was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC; K18-Hep-7d VS K18-NaTPP-7d p\u0026thinsp;=\u0026thinsp;0.0182). K18-NaTPP showed a higher fluorescence intensity, almost double the one of K18-Hep. After 10 days, the growing phase ended, and the fluorescence intensity remained constant, showing a slight decrease after 14 days, probably caused by the formation of very insoluble aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The major differences in fibrillation kinetics were observed during the growth phase, between 4 and 7 days. Therefore, to explore the effects of the NaTPP on the formation and morphology of K18 fibrils, these two time points were further investigated by scanning transmission electron microscopy (STEM). K18 exposed to heparin (1:1 in molar concentrations) showed great difference after 4 and 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD-F). Population distribution of the K18-Hep sample after 4 days at 37\u0026deg;C resulted in a scarce amount of fibrils (1.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13%), while after 7 days the population of fibrils was richer (19.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4.25%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE; K18-Hep-4d VS K18-Hep-7d p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001). The differences between these two samples concerned also the length of the fibrils. In fact, after 4 days of exposure to heparin, K18 presented higher fibril variability, whereas the protein showed multiple aggregates with a more consistent and final fibril length after 7 days (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF; K18-Hep-4d VS K18-Hep-7d p\u0026thinsp;=\u0026thinsp;0.0093). This behavior was similar for K18-NaTPP samples. On average, these fibrils were longer than K18-Hep fibrils (K18-NaTPP\u0026thinsp;\u0026gt;\u0026thinsp;800 nm, K18-Hep\u0026thinsp;\u0026lt;\u0026thinsp;400 nm, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF). However, after 4 days at 37\u0026deg;C, the fibrils still showed oscillations in dimension, whilst after 7 days, the length variability was lower, although still present (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eF; K18-NaTPP-4d VS K18-NaTPP-7d p=?). The population distribution of K18-NaTPP fibrils after 4 days (2.94\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72%) was similar to the same sample after 7 days (3.28\u0026thinsp;\u0026plusmn;\u0026thinsp;1.25%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eE). These data indicate that K18 exposed to NaTPP produces longer fibrils than heparin, showing fibril maturity after 7 days.\u003c/p\u003e\n\u003ch3\u003eMolecular Dynamics Simulations Reveal NaTPP-Induced Aggregation Mechanisms of K18 Tau Protein\u003c/h3\u003e\n\u003cp\u003eWith the aim of investigating the equilibrium conformational exploration of K18, we performed a one-microsecond molecular dynamics simulation of K18 in the presence of NaTPP. As control we also performed two additional one-microsecond molecular dynamics simulations, considering K18 protein alone in water, and K18 in the presence of sodium monophosphate (Na\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e)(NaP).\u003c/p\u003e \u003cp\u003eA set of preliminary analyses (Root Mean Square Deviation (RMSD), radius of gyration, and contact analysis, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA-B-C) for all three simulated systems highlighted that the K18 protein adopts a more compact conformation, regardless of the presence of ligands (see Supplementary Material for more details).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eTo investigate the dynamic-structural stability of the solvent-exposed regions involved in molecular binding, in this specific case, we leverage the experimental knowledge of the system (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Therefore, we selected the three interacting glycines (GGG patch: residues 333, 334, and 335) from PDB 5O3L, which are central residues of the interface. A cartoon representation of this structure is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE (left), where the three glycines in question are highlighted in red.\u003c/p\u003e \u003cp\u003eWe calculated the area of molecular iso-electron density surfaces formed by the three glycines during the molecular dynamics simulation for the three molecular systems separately, with the idea that the larger the area of the considered surface portion, the greater the possibility of interaction.\u003c/p\u003e \u003cp\u003eFor the two systems that did not form aggregates (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD, free K18, top trace, and K18 with NaP, middle trace), the solvent-exposed area of residues involved in the fibril interface fluctuated significantly throughout the simulation, transitioning from high exposure values (in black), potentially favorable to binding, to low solvent exposure values (in red), indicative of conformations that theoretically hinder binding.\u003c/p\u003e \u003cp\u003eInterestingly, when K18 interacted with NaTPP, after approximately 500 ns, it began to explore conformations that did not present a marked structural stability compared to the first part of the simulation (see RMSD and radius of gyration analysis) but maintained a highly stable area value in the interface region. The evidence of this signal in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eD (bottom trace) is of great interest, with an average area value of 121\u0026Aring;\u0026sup2;, with a standard deviation of 11 \u0026Aring;\u0026sup2;. In contrast, during the first half of the simulation, the same system exhibited a more pronounced variability in surface area, with an average of 113 \u0026Aring;\u0026sup2; and a standard deviation of 24 \u0026Aring;\u0026sup2;. A structural visualization of the surface areas related to the three glycines is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, where we show a snapshot of the trajectory in which the three glycines were partially buried (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, middle) and one, belonging to the second half of the simulation of K18 in the presence of NaTPP, in which the three glycines are markedly exposed (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eE, right).\u003c/p\u003e \u003cp\u003eThe stabilization of the solvent-exposed area of ​​residues involved in the fibril interface suggests the importance of stabilizing not only the entire protein structure (probably a necessary condition) but also the binding region (a necessary and sufficient condition).\u003c/p\u003e \u003cp\u003eAdditionally, based on the ligand-protein distance, we defined the residues in contact with the NaTPP at each frame of the simulation to calculate the relative contact frequency of each residue separately for the first part and the second part of the molecular dynamics simulation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, left).\u003c/p\u003e \u003cp\u003eIn general, the contacts of the K18 residues interacting with NaTPP are largely conserved. However, in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, right, we highlighted in green the residues that have significantly increased the frequency of contact with the ligand in the second half of the simulation, compared to the first part of the trajectory (55I, 56K, 65Q, 66I, and 81G). On the contrary, in yellow, we reported the residues with many contacts in the first part and a loss of contacts in the second half of the trajectory.\u003c/p\u003e \u003cp\u003eThe initial residues in the N-terminus of K18 also became consistently interacting in the second half of the simulation, indicating that the new rearrangement of the protein configuration could also be induced by a new interaction between the ligand and the N-terminal of K18.\u003c/p\u003e \u003cp\u003eDuring the first half of the simulation, when the area of the GGG patch involved in the fibril interface was variable and generally less exposed, this region frequently interacted with two sets of residues, one centered around 55I and 56K residues, and the other centered around 65Q and 66I residues. At the end of the first half of the simulation, a significant conformational change occurred in K18, as shown in the previous analysis.\u003c/p\u003e \u003cp\u003eIn the second half of the simulation, the pairs of residues 55I-56K and 65Q-66I, which formed the basis of a loop created in K18, came closer together and interacted with the ligand (thus moving away from the three glycines belonging to the fibril interface). During the first part of the simulation, these residues were not at the base of the loop and were less constrained. On the contrary, the new configuration restricted the length of this region free to move, making it insufficient to prevent the three glycines from interacting with the solvent. In Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eF, snapshots of the simulation are shown, depicting the contact between residues 55I, K56, 65Q, and 66I (in green) and the three glycines (in red) during the first phase and their involvement in binding with the NaTPP during the second half of the simulation.\u003c/p\u003e \u003cp\u003eThis conformation remained locally stable and ensured the constant exposure of the GGG patch, as evident from the molecular surface area plot.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eShort-Term Cellular Responses to K18 Fibrils: Increased Endogenous Tau and Autophagic Activity\u003c/h2\u003e \u003cp\u003eTo study the impact of K18 fibrils on retinal neurons, we treated DIV 30 human iPSC-derived retinal cultures with 5 \u0026micro;M of K18 fibrils for 24 hours after confirming K18 fibril internalization and toxicity (Supplementary Figure S3, Scheme S3D). Increased endogenous tau protein expression was observed with K18-Hep-4d, K18-Hep-7d, and K18-NaTPP-4d treatments but not with K18-NaTPP-7d. This was verified using an anti-total tau (HT7) antibody that recognizes the \"PPGQK\" epitope of human tau, absent in K18 (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA), thus detecting only endogenous tau (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). Similar results were found with the cytoskeletal marker MAP2 (Supplementary Fig.\u0026nbsp;4).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eInterestingly, K18-NaTPP-4d treatment led to the highest expression of endogenous tau protein compared to other samples (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). This increase in tau was not associated with changes in β-III Tubulin (TUJ1) (Supplementary Fig. S4), supporting the hypothesis that exogenous fibrils trigger a stress response, leading to cellular upregulation of endogenous tau (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e16\u003c/span\u003e), (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e52\u003c/span\u003e). We also observed increased p62/SQSTM1 puncta following K18 fibril treatment (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Notably, p62 puncta were significantly more abundant in cultures treated with K18-NaTPP-4d than those treated with K18-NaTPP-7d or K18-Hep-4d/7d (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). Additionally, p62 puncta colocalized with exogenous K18 fibrils, with the strongest colocalization observed in K18-Hep-4d and K18-NaTPP-4d treatments (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eE-\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eF). These results indicate an increase in autophagic flux after 24 hours, aligning with p62 neuroprotective role in reducing oligomeric tau. However, we did not observe Cleaved-caspase3 activation, indicating that short-term K18 exposure does not induce apoptosis (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG-H).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eLong-Term Neurodegenerative Effects of K18 Fibrils: Cytoskeleton Alteration and Apoptotic Pathway Activation\u003c/h3\u003e\n\u003cp\u003eWe assessed the impact of K18 fibril treatment on neuronal dysfunction and neurodegeneration two weeks after incubation (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). A live-dead assay revealed a notable reduction in live cells, with a 40% decrease in cultures internalizing K18-Hep-4d and K18-Hep-7d fibrils, a 45% decrease after K18-NaTPP-4d treatment, and a 30% decrease following K18-NaTPP-7d exposure (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB). Immunostaining showed a significant decrease in the neurite marker TUJ1 area in cultures treated with K18-Hep-7d, K18-NaTPP-4d, and K18-NaTPP-7d fibrils compared to controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD left). Additionally, fibril-treated cultures exhibited smaller TUJ1 fragments (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eD middle and right; Supplementary Fig. S5). These findings indicate that internalized fibrils gradually disrupt the neuronal cytoskeleton, with more severe effects observed in K18-NaTPP fibril-treated cultures.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn this model, fibril treatment also led to the formation of hyperphosphorylated pathological tau (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e16\u003c/span\u003e). We measured tau phosphorylation at the Ser-202, Thr-205 (AT8) epitopes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC) and found a significant increase in phosphorylated tau within neurites of treated cells compared to untreated ones (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC, zoomed panel; Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eE). We then explored the role of p62/SQSTM1 in processing phosphorylated tau. Immunofluorescence analysis revealed a marked increase in p62/SQSTM1 puncta in cultures treated with K18-Hep fibrils, but not in those treated with K18-NaTPP fibrils (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eF-\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eG). Additionally, we observed a significant activation of the apoptotic pathway two weeks post-treatment, indicated by an increased Cleaved Caspase-3 signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eH). Collectively, these findings suggest that K18 fibril treatment induces long-term neurite degeneration and neuronal loss, which contribute to neurodegeneration and ultimately lead to cell death.\u003c/p\u003e"},{"header":"DISCUSSION","content":"\u003cp\u003eIn this study, we report the differential effects of K18 tau fibrils, induced by heparin and NaTPP, on human iPSC-derived retinal neurons (\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e51\u003c/span\u003e) over short-term and long-term exposures. NaTPP and heparin impact fibrillation kinetics (\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e29\u003c/span\u003e) and morphology differently, leading to distinct structural and cellular responses. Notably, NaTPP-induced fibrils showed a more rapid and extensive aggregation process compared to heparin, and produced longer, more stable fibrils over seven days. These distinctions in fibrillation dynamics translate into varied cellular effects, shedding light on the mechanisms by which different fibril structures interact with neuronal cells and contribute to neurodegeneration.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eShort-Term Cellular Responses: Tau Induction and Autophagy Activation\u003c/h2\u003e \u003cp\u003eOur findings suggest that K18 fibrils induce an acute stress response in retinal neurons, leading to increased expression of endogenous tau protein. This effect was most pronounced in cultures treated with K18-NaTPP-4d, which showed the highest tau upregulation among the fibril types. The selective increase in tau, confirmed via anti-tau (HT7) immunostaining, implies that exogenous fibrils may stimulate a compensatory cellular mechanism aimed at buffering against tau aggregation stress. Furthermore, K18-NaTPP-4d also caused a notable rise in autophagic flux, as indicated by the substantial increase in p62/SQSTM1 puncta, colocalized with exogenous K18 fibrils, suggesting that autophagy serves a neuroprotective role by targeting tau aggregates for degradation. Importantly, there was no evidence of Cleaved Caspase-3 activation during this short-term exposure, indicating that while K18 fibrils impose cellular stress, they do not immediately trigger apoptosis within 24 hours.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eLong-Term Neurodegenerative Effects: Cytoskeleton Disruption and Apoptosis\u003c/h2\u003e \u003cp\u003eProlonged exposure to K18 fibrils led to marked neurodegenerative outcomes, highlighting the cumulative impact of sustained fibril presence on neuronal integrity. Two weeks post-treatment, we observed a significant reduction in cell viability, with the most severe decreases in cultures treated with K18-Hep-4d, K18-Hep-7d, and K18-NaTPP-4d fibrils. This decline in live cells, accompanied by a reduction in TUJ1-labeled neurite area, indicates that prolonged fibril internalization progressively disrupts the neuronal cytoskeleton. Furthermore, fibril-treated cells exhibited hyperphosphorylated tau at the AT8 epitope, a hallmark of tauopathy, suggesting that continuous fibril exposure fosters pathogenic tau modifications that contribute to structural degeneration.\u003c/p\u003e \u003cp\u003eInterestingly, long-term exposure also differentially impacted p62/SQSTM1 activity across fibril types. While K18-Hep fibrils prompted an increase in p62/SQSTM1 puncta, this was not observed with K18-NaTPP fibrils. This suggests that K18-Hep fibrils may induce a stronger autophagic response to manage phosphorylated tau, whereas K18-NaTPP fibrils may engage alternative pathways or mechanisms. The eventual activation of the apoptotic pathway, as evidenced by Cleaved Caspase-3 staining, underscores a shift from protective responses to neurodegenerative processes, marking a tipping point where cellular defenses become insufficient to counteract fibril-induced toxicity.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eMolecular Dynamics Insights: Structural Basis of NaTPP-Induced Aggregation\u003c/h2\u003e \u003cp\u003eThe K18-NaTPP-7d fibrils appear to have adopted a helicoidal mature structure, contrasting with the shorter, entangled aggregates observed in K18-Hep fibrils and K18-NaTPP-4d, which may be still in the protofibril phase (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e54\u003c/span\u003e), (\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e55\u003c/span\u003e), (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e56\u003c/span\u003e). Over time, however, both NaTPP- and heparin-induced fibrils led to tau pathology and neuronal death. In line with the hypothesis that mature fibrils, rather than intermediates, are less toxic, these differences in fibril behavior may be related to variations in their size and morphology. K18-Hep-4d fibrils were smaller and more heterogeneous, while K18-Hep-7d fibrils formed longer, more numerous structures, though their length did not exceed 400 nm. Conversely, K18-NaTPP-4d fibrils were more homogeneous and over 800 nm long, resembling the morphology of 7-day fibrils. Alongside longer polyphosphates, our results indicated that even short-chain polyphosphates like NaTPP can reduce the lag phase of fibril formation and promote elongation (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e47\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e53\u003c/span\u003e). Molecular dynamics simulations further confirmed this hypothesis. The root mean square deviation (RMSD) and radius of gyration indicated distinct conformational behaviors of Tau protein in water, phosphate buffer, and NaTPP. In this condition, K18 exhibited rapid compaction early in the simulation, a feature absent in the other systems, which likely indicates an aggregated conformation. A key finding was the behavior of the GGG patch (residues 333\u0026ndash;335), crucial for tau fibril formation. In the K18-NaTPP system, the GGG patch remained solvent-exposed, suggesting that NaTPP stabilizes the aggregation-prone conformation of K18. Protein-ligand interactions also played a role, with specific residues (55I, 56K, 65Q, 66I, 81G) forming stable contacts with NaTPP, further stabilizing the aggregation-prone state by maintaining the exposure of the GGG patch. Although the mechanism that induces tau fibril formation is highly complex and likely results from the interaction of multiple concomitant factors, our study highlighted an unexplored role of NaTPP, whose presence is physiologically relevant in neurons (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e29\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe physiopathological relevance of NaTPP-induced tau fibrillation lies in its ability to mimic endogenous polyphosphates (polyP), which significantly influence neuronal function and excitability but also participate in pathological tau aggregation. NaTPP-induced tau fibrillation highlights the dual nature of polyP in neuronal homeostasis: while polyP modulates ion channels, regulates calcium signaling, and offers neuroprotective effects, it also promotes tau aggregation under certain conditions (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e57\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e58\u003c/span\u003e). This aggregation contributes to neurotoxicity by enhancing pathological tau structures associated with neurodegenerative processes, suggesting that while polyP plays a critical role in neuronal health, its impact on tau fibrillation may drive toxic outcomes in disease states.\u003c/p\u003e \u003cp\u003eOverall, our study demonstrated that the short-chain NaTPP plays a key role in tau folding into tangles due to molecular interactions with specific residues of K18 and in the development of pathological cellular responses in iPSC cells, providing a basis for further investigation into their potential role in neurodegenerative disease.\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Alzheimer\u0026apos;s disease\u003c/p\u003e\n\u003cp\u003eA\u0026beta;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026beta;-amyloid\u003c/p\u003e\n\u003cp\u003eDIV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Day in vitro\u003c/p\u003e\n\u003cp\u003eFDA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fluorescein diacetate\u003c/p\u003e\n\u003cp\u003eFOV\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Field of view\u003c/p\u003e\n\u003cp\u003eFTD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Fronto temporal dementia\u003c/p\u003e\n\u003cp\u003eGGG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;3 interacting glycines\u003c/p\u003e\n\u003cp\u003ehiPSC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Human induced pluripotent stem cells\u003c/p\u003e\n\u003cp\u003eHumAfFt-BT1\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;BT1-loaded ferritin\u003c/p\u003e\n\u003cp\u003eiPSC\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Induced pluripotent stem cells\u003c/p\u003e\n\u003cp\u003eIPTG\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Isopropyl \u0026beta;-D-1-tiogalattopiranoside\u003c/p\u003e\n\u003cp\u003eK18\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Microtubule-binding domain\u003c/p\u003e\n\u003cp\u003eMTBR\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Microtubule-binding repeat region\u003c/p\u003e\n\u003cp\u003eNaP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Sodium monophosphate\u003c/p\u003e\n\u003cp\u003eNaTPP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Sodium tripolyphosphate\u003c/p\u003e\n\u003cp\u003eNES\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;HEPES-buffered external solution\u003c/p\u003e\n\u003cp\u003eNFT\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Neurofibrillary tangles\u003c/p\u003e\n\u003cp\u003ePDF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Probability density functions\u003c/p\u003e\n\u003cp\u003ePFA\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Paraformaldehyde\u003c/p\u003e\n\u003cp\u003ePHF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Paired helical filaments\u003c/p\u003e\n\u003cp\u003ePI\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Propidium Iodide\u003c/p\u003e\n\u003cp\u003ePolyPs\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Polyphosphates\u003c/p\u003e\n\u003cp\u003eRMSD\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Root mean square deviation\u003c/p\u003e\n\u003cp\u003eSF\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Straight filaments\u003c/p\u003e\n\u003cp\u003eSTEM\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;Scanning transmission electron microscopy\u003c/p\u003e\n\u003cp\u003eTCEP\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Tris(2-carboxyethyl) phosphine hydrochloride\u003c/p\u003e\n\u003cp\u003eThT \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;Thioflavin T\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAVAILABILITY OF DATA AND MATERIALS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eFor the analysis of imaging data, we utilized custom code developed in MathWorks Matlab (version 2016b), which is available upon request.\u003c/p\u003e\n\u003cp\u003eAll the protein structures used in this paper are available in the Protein Data Bank. The Data generated with molecular dynamics and molecular docking will be available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eACKNOWLEDGEMENTS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors wish to thank the Center for Life Nano and Neuroscience Imaging Facility, Istituto Italiano di Tecnologia.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFUNDING\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by MUR PRIN 2022 (CUP: \u0026nbsp; 2022CFP7RF, to SDA and PB, EM). This work was supported by Regione LAZIO Lazio-Innova; POR FESR Lazio 2014-2020 (A0375-2020-36549, CUP: B85F20003340002 to PB). This research was also funded by the D-Tails-IIT Joint Lab (to SDA, YG, AB), the Regione Lazio FSE 2014\u0026ndash;2020 (19036AP000000019 and A0112E0073) grants (to SDA), Sapienza University grants (RM118163E0297F84, PH12017270934C3C, MA32117A7B698029 and RM1231889BB77735to SDA; RM123188F763A9D5 to PB), and Fondazione Istituto Italiano di Tecnologia (to LM). LM was also supported by the PhD program in Life Science at Sapienza University in Rome. SDA was also supported by Progetto ECS 0000024 Rome Technopole, - CUP B83C22002820006, PNRR Missione 4 Componente 2 Investimento 1.5, finanziato dall\u0026rsquo;Unione europea \u0026ndash; NextGenerationEU. SDA was also supported by the Italian Ministry of Health - Alternative Methods to Animal Testing Grant 2023 (NEURO-3R). AB, GR and PB were also supported by \u0026nbsp;Project \u0026quot;National Center for Gene Therapy and Drugs based on RNA Technology\u0026quot; (CN00000041) financed by NextGenerationEU PNRR MUR\u0026mdash;M4C2\u0026mdash;Action 1.4-Call \u0026quot;Potenziamento strutture di ricerca e creazione di \u0026quot;campioni nazionali di R\u0026amp;S\u0026quot; (CUP J33C22001130001). LB was supported by Progetto Avvio alla Ricerca 2022 (University \u0026ldquo;La Sapienza,\u0026rdquo; Rome, Italy).\u0026nbsp;The research leading to these results was also supported by the European Research Council through its Synergy grant program, project ASTRA (grant agreement No 855923) and by the European Innovation Council through its Pathfinder Open Programme, project ivBM-4PAP (grant agreement No 101098989).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eETHICS DECLARATIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe use of hiPSC has been approved by the Ethical Committee for Translational Research (CERT) Sapienza University (Aut. n. 5/2022).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe funders had no role in the study design, in the collection, analysis, or interpretation of data, in the writing of the manuscript, or in the decision to publish the results.\u003c/p\u003e\n\u003cp\u003eYG is employed by D-Tails s.r.l.; SDA is a scientific advisor of D-Tails s.r.l. The remaining authors declare that the research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAUTHOR CONTRIBUTIONS\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLB performed biochemical experiments and related chemical analysis; YG performed retinal differentiation experiments \u0026nbsp;(confocal microscopy, immunofluorescence, internalization) and related analysis; LM performed retinal differentiation experiments and molecular and cellular characterization; SG wrote the MATLAB code and performed quantification of confocal acquisitions; FM performed STEM acquisition; MVF performed spectral analysis; ST and LD performed EM analysis; \u0026nbsp; AB and PB designed the biochemical experimental strategy; SDA designed biological experimental strategy; GR and EM designed the computational approach; EM performed the computational analysis; AB, GR, PB, EM and SDA conceived the study; PB, EM and SDA designed the experiments, interpreted the results, and wrote the manuscript. The manuscript was written through the contributions of all authors. All authors have approved the final version of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eBinder LI, Frankfurter A, Rebhun LI. The distribution of tau in the mammalian central nervous system. The Journal of cell biology. 1985;101(4):1371\u0026ndash;8. \u003c/li\u003e\n\u003cli\u003eChang CW, Shao E, Mucke L. Tau: Enabler of diverse brain disorders and target of rapidly evolving therapeutic strategies. Science. 2021;371(6532):eabb8255. \u003c/li\u003e\n\u003cli\u003eGoedert M, Jakes R, Spillantini MG, Hasegawa M, Smith MJ, Crowther RA. Assembly of microtubule-associated protein tau into Alzheimer-like filaments induced by sulphated glycosaminoglycans. Nature;383(6600):550\u0026ndash;3. \u003c/li\u003e\n\u003cli\u003eHern\u0026aacute;ndez F, Ferrer I, P\u0026eacute;rez M, Zabala JC, Del Rio JA, Avila J. Tau Aggregation. 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Comparison of simple potential functions for simulating liquid water. The Journal of Chemical Physics. 1983;79(2):926\u0026ndash;35. \u003c/li\u003e\n\u003cli\u003eCremers CM, Knoefler D, Gates S, Martin N, Dahl JU, Lempart J, et al. Polyphosphate: A Conserved Modifier of Amyloidogenic Processes. Molecular Cell. 2016;63(5):768\u0026ndash;80. \u003c/li\u003e\n\u003cli\u003eMurphy RM. Kinetics of amyloid formation and membrane interaction with amyloidogenic proteins. Biochimica et Biophysica Acta (BBA) - Biomembranes. 2007;1768(8):1923\u0026ndash;34. \u003c/li\u003e\n\u003cli\u003eKumar AV, Mills J, Lapierre LR. Selective Autophagy Receptor p62/SQSTM1, a Pivotal Player in Stress and Aging. Front Cell Dev Biol. 2022;10:793328.\u003c/li\u003e\n\u003cli\u003eMaiolino M, O\u0026rsquo;Neill N, Lariccia V, Amoroso S, Sylantyev S, Angelova PR, Abramov AY. Inorganic Polyphosphate Regulates AMPA and NMDA Receptors and Protects Against Glutamate Excitotoxicity via Activation of P2Y Receptors. Journal of Neuroscience 2019;39(31):6038-6048.\u003c/li\u003e\n\u003cli\u003eStotz SC, Scott LO, Drummond-Main C, Avchalumov Y, Girotto F, Davidsen J, G\u0026oacute;mez-G\u0026aacute;rcia MR, Rho JM, Pavlov EV, Colicos MA. Inorganic polyphosphate regulates neuronal excitability through modulation of voltage-gated channels. Mol Brain (2014);7(42):1756-6606.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5409787/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5409787/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe onset of Alzheimer\u0026rsquo;s Disease and Frontotemporal Dementia is closely associated with the aggregation of tau, a multifunctional protein essential for neuronal stability and function. Given the role of tau aggregation in neurodegeneration, understanding the mechanisms behind its fibrils formation is crucial for developing therapeutic interventions to halt or reverse disease progression. However, the structural complexity and diverse aggregation pathways of tau present significant challenges, requiring comprehensive experimental studies. In this research, we demonstrate that short-chain polyphosphates, specifically sodium tripolyphosphate (NaTPP), effectively induce tau fibril formation \u003cem\u003ein vitro\u003c/em\u003e using the microtubule-binding domain fragment (K18). NaTPP-induced fibrils display unique structural characteristics and aggregation kinetics compared to those induced by heparin, indicating distinct pathogenic pathways.\u003c/p\u003e \u003cp\u003eThrough molecular dynamics simulations, we show that NaTPP promotes aggregation by exposing key residues necessary for fibril formation, which remain concealed under non-aggregating conditions. This interaction drives tau into an aggregation-prone state, revealing a novel mechanism. Furthermore, our study indicates that human pluripotent stem cell-derived retinal neurons internalize NaTPP-induced fibrils within 24 hours, pointing to a potential pathway for tau spread in neurodegeneration.\u003c/p\u003e \u003cp\u003eTo explore the translational implications of NaTPP-induced fibrils, we assessed their long-term effects on cellular viability, tubulin integrity, and stress responses in retinal neuron cultures. Compared to heparin, NaTPP promoted fewer but longer fibrils with initially low cytotoxicity but induced a stress response marked by increased endogenous tau and p62/SQSTM1 expression. Prolonged exposure to NaTPP-induced oligomers significantly increased cytotoxicity, leading to tubulin fragmentation, altered caspase activity, and elevated levels of phosphorylated pathological tau. These findings align with a neurodegenerative phenotype, highlighting the relevance of polyphosphates in tau pathology.\u003c/p\u003e \u003cp\u003eOverall, this research enhances our understanding of the role of polyphosphate in tau aggregation, linking it to key cellular pathways in neurodegeneration.\u003c/p\u003e","manuscriptTitle":"Short-Chain Polyphosphates Induce Tau Fibrillation and Neurotoxicity in Human iPSC-Derived Retinal Neurons","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-02 04:37:47","doi":"10.21203/rs.3.rs-5409787/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"revise","date":"2024-12-19T14:31:06+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-12-03T11:16:47+00:00","index":2,"fulltext":"This content is not available."},{"type":"editorInvitedReview","content":"This content is not available.","date":"2024-11-25T10:05:28+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-11-19T13:24:14+00:00","index":2,"fulltext":"This content is not available."},{"type":"reviewerAgreed","content":"This content is not available.","date":"2024-11-19T01:44:53+00:00","index":1,"fulltext":"This content is not available."},{"type":"reviewersInvited","content":"","date":"2024-11-15T16:29:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-11-08T12:10:11+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-11-07T12:08:11+00:00","index":"","fulltext":""},{"type":"submitted","content":"Cell Death \u0026 Disease","date":"2024-11-07T12:08:10+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"cell-death-and-disease","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"cddis","sideBox":"Learn more about [Cell Death \u0026 Disease](http://www.nature.com/cddis/)","snPcode":"41419","submissionUrl":"https://mts-cddis.nature.com/cgi-bin/main.plex","title":"Cell Death \u0026 Disease","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"04153527-4ca6-48bf-a812-09618371c02f","owner":[],"postedDate":"December 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":40328638,"name":"Biological sciences/Neuroscience/Stem cells in the nervous system"},{"id":40328639,"name":"Biological sciences/Biochemistry/Proteins"}],"tags":[],"updatedAt":"2025-05-10T07:08:32+00:00","versionOfRecord":{"articleIdentity":"rs-5409787","link":"https://doi.org/10.1038/s41419-025-07662-5","journal":{"identity":"cell-death-and-disease","isVorOnly":false,"title":"Cell Death \u0026 Disease"},"publishedOn":"2025-05-09 04:00:00","publishedOnDateReadable":"May 9th, 2025"},"versionCreatedAt":"2024-12-02 04:37:47","video":"","vorDoi":"10.1038/s41419-025-07662-5","vorDoiUrl":"https://doi.org/10.1038/s41419-025-07662-5","workflowStages":[]},"version":"v1","identity":"rs-5409787","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5409787","identity":"rs-5409787","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}